Modification of plant metabolism

ABSTRACT

A transgenic plant is prepared by a method n which a plant cell is transformed with a chimaeric gene comprising a promoter and a gene encoding a polypeptide which displays the activity of an enzyme which regulates the amount of a metabolic intermediate in glycolysis or in a pathway for the synthesis or degradation of starch, sucrose or reducing sugar from a glycotyltic intermediate. In stored potatoes the subject of the invention an increased level of phosphofructokinase results in reduced accumulations of sugars in the tubers.

The present invention relates to transgenic plants and theirpreparation.

Phosphofrucktokinase (PFK:EC 2.7.1.11) is widely regarded as a keyregulatory enzyme controlling the entry of carbon into glycolysis.Glycolysis, especially in plant cells, serves to supply both respiratorycarbon for energy production and intermediates for other metabolicpathways. The potato tuber contains four forms of PFK (Kruger et al,Arch. Biochem. Biophys. 267, 690-700) and pyrophosphatefructose-6-phosphate phosphotransferase (PFP:EC 2.7.1.90) which cancatalyse the conversion of fructose-6-phosphate tofructose-1,6-bisphosphate. PFK is present in both the cytosol and theamyloplast while PFP is only known to occur in the cytosol.

It has previously been thought that PFK alone controls the totalglycolytic flux. However, we have now found that this is not the case.We introduced additional PFK into potato plants by genetic manipulation.Our results indicate that a substantial increase in PFK activity did notsubstantially alter flux through glycolysis but changed the pool sizesof intermediates. The results indicate that regulation of glycolyticflux may be achieved not only at the entry of carbon into the pathwaybut also exit from it. This finding has general applicability.

Accordingly, the present invention provides a process for thepreparation of a transgenic plant, which method comprises:

-   -   (i) transforming a plant cell with a chimaeric gene        comprising (a) a suitable promoter and (b) a coding sequence the        product of which causes modification of the amount of metabolic        intermediate in glycolysis or in a pathway for the synthesis or        degradation of starch, sucrose or reducing sugar; and    -   (ii) regenerating a plant from the transformed cell.

The invention also provides the chimaeric gene. A vector suitable foruse in the present process comprises the chimaeric gene such that thechimaeric gene is capable of being expressed in a plant cell transformedwith a vector. A plant cell according to the invention thereforeharbours the chimaeric gene such that the chimaeric gene is capable ofbeing expressed therein.

A transgenic plant can therefore be obtained which harbours in its cellsthe chimaeric gene such that the chimaeric gene is capable of beingexpressed in the cells of the plant. Seed or other propagules can beobtained from the transgenic plant and used to grow further plantsstably transformed with the chimaeric gene.

The invention enables plant metabolism to be altered in a glycolyticpathway or in a pathway for the synthesis or degradation of starch,sucrose or a reducing sugar such as glucose or fructose. It enables theaccumulation of pathway metabolites to be altered. Several applicablepathways are shown in FIG. 1 of the accompanying drawings. The inventionis particularly applicable to potatoes. It had been expected that theintroduction and expression of additional PFK into potato tuber cellswould cause a high flux in the glycolytic pathway. Furthermore, if thisgene had been introduced and expressed in the whole plant, it would nothave been unreasonable to have expected that the plant would have died.In the event, though, it was surprising to find that after theintroduction and expression of the PFK gene the plant did not die andthe flux in the glycolysis metabolism pathway was not increased.

The storage of potato tubers in low temperature storage conditionsnormally results in less PFK activity. This, it is believed, leads to anincreased production in the potato tubers of sucrose and reducingsugars. The accumulation of these sugars in the potato tubers presents asignificant problem to processors of potatoes. For example, producers ofcrisps and chips (otherwise known respectively as potato chips andFrench fries) have found that the presence of an increased level ofsugars tends to cause an undue browning of the products during thefrying process.

When potato tubers of the subject invention are stored at lowtemperatures, the increased amount of PFK present therein ensures acontinued flux into the glycolysis metabolism pathway. This in turnmeans that the flux level in the sucrose synthesis pathway is lower thanhas heretofore been the case with stored potato tubers. Thussignificantly reduced levels of sucrose and reducing sugars accumulatesin the stored tubers.

In the invention, a chimaeric gene is constructed which comprises (a) asuitable promoter operably linked to (b) a coding sequence the productof which causes modification of the amount of a metabolic intermediatein glycolysis or in a pathway for the synthesis or degradation ofstarch, sucrose or reducing sugar. The chimaeric gene may be constructedin any convenient fashion. The coding sequence is provided such that itis expressible in plant cells.

The promoter (a) should be capable of expressing the coding sequence ofinterest in a plant. The promoter may be a promoter capable of directingexpression in a particular tissue of a plant and/or at particular stagesof development. The promoter may be heterologous or homologous to theplant. A suitable promoter may be the 35S cauliflower mosaic viruspromoter, a nopaline synthase or octopine synthase promoter, a patatinpromoter or a small sub-unit of rubisco. A promoter from tubers, e.g.patatin, is preferred for directing expression in potatoes, inparticular potato tubers. A suitable promoter may be, for example, aconstitutive promoter or a tissue-specific promoter.

The coding sequence (b) can encode an enzyme which regulates the amountof a metabolic intermediate of a specific pathway. The pathway may bethe glycolytic pathway. Glycolysis is the sequence of reactions whichconverts glucose to pyruvate with concomitant production of ATP or NADHand is also termed the Embden-Meyerhof-Parnas pathway.

Sucrose consists of glucose and fructose coupled via an alpha 1-2O-glycosidic bond. Pathways of sucrose synthesis therefore involveenzyme steps that produce suitable intermediates to form this linkage.Starch is a polymer which consists mainly of alpha 1-4 linked glucosewith varying amounts of 1-6 linked glucose. Thus pathways of starchsynthesis involve steps that produce suitable intermediates to form thispolymer.

A coding sequence is selected which when expressed in plant cells willincrease or decrease the metabolism of a pathway mentioned above. Thecoding sequence (b) may encode for a pathway enzyme or an activemodified form of a pathway enzyme, for example a truncated pathwayenzyme. The pathway enzyme may be, for example, PFK (EC 2.7.1.11),pyruvate kinase (PK) (EC 2.7.1.40), acid invertase (EC 3.2.1.26), starchsynthase (EC 2.4.1.21), adenine diphosphoglucose pyrophosphorylase (EC3.6.1.21), sucrose synthase (EC 2.4.1.13), 6-phosphofructokinase(pyrophosphate) (EC 2.7.1.90) or sucrose phosphate synthetase (SPS) (EC2.4.1.14).

The coding sequence may be from a plant gene or a non-plant gene such asa microbial gene. It may be from a bacterial gene, for example a genefrom E. coli, or a yeast gene, for example Saccharomyces cerevisiae. Inparticular, a PFK coding sequence may be provided by the pfkA gene fromE. coli or by a pfk gene from Solanum tuberosum. An acid invertasecoding sequence may be provided from Saccharomyces cerevisiae.

Plant cells can be transformed with the chimaeric gene by direct DNAuptake, typically by way of a DNA fragment comprising the chimaericgene. Alternatively, there may be used a vector incorporating thechimaeric gene. The chimaeric gene typically includes transcriptionalcontrol sequences, for example a promoter as above, and translationalinitiation and/or termination sequences. Plant terminator andpolyadenylation sequences may be present. A vector typically contains toa region which enables the chimaeric gene to be transferred to andstably integrated in the plant cell genome.

The vector is therefore typically provided with transcriptionalregulatory sequences and/or, if not present at the 3′-end of the codingsequence of the gene, a stop codon. A DNA fragment may therefore alsoincorporate a terminator sequence and other sequences which are capableof enabling the gene to be expressed in plant cells. An enhancer orother element able to increase or decrease levels of expression obtainedin particular parts of a plant or under certain conditions, may beprovided in the DNA fragment and/or vector. The vector is also typicallyprovided with an antibiotic resistance gene which confers resistance ontransformed plant cells, allowing transformed cells, tissues and plantsto be selected by growth on appropriate media containing the antibiotic.

Transformed plant cells can be selected by growth in an appropriatemedium. Plant tissue can therefore be obtained comprising a plant cellwhich harbours a gene encoding an enzyme under the control of apromoter, for example in the plant cell genome. The gene is thereforeexpressible in the plant cell. Plants can then be regenerated whichinclude the gene and the promoter in their cells, for example integratedin the plant cell genome such that the gene can be expressed. Theregenerated plants can be reproduced and, for example, seed obtained.

A preferred way of transforming a plant cell is to use Agrobacteriumtumefaciens containing a vector comprising a chimaeric gene as above. Ahybrid plasmid vector may therefore be employed which comprises:

-   -   (a) a chimaeric gene containing regulatory elements capable of        enabling the gene to be expressed when integrated in the genome        of a plant cell;    -   (b) at least one DNA sequence which delineates the DNA to be        integrated into the plant genome; and    -   (c) a DNA sequence which enables this DNA to be transferred to        the plant genome.

Typically the DNA to be integrated into the plant cell genome isdelineated by the T-DNA border sequences of a Ti-plasmid. If only oneborder sequence is present, it is preferably the right border sequence.The DNA sequence which enables the DNA to be transferred to the plantcell genome is generally the virulence (vir) region of a Ti-plasmid.

The gene coding for the enzyme and its transcriptional and translationalcontrol elements can therefore be provided between the T-DNA borders ofa Ti-plasmid. The plasmid may be a disarmed Ti-plasmid from which thegenes for tumorigenicity have been deleted. The gene and itstranscriptional control elements can, however, be provided between T-DNAborders in a binary vector in trans with a Ti-plasmid with a vir region.Such a binary vector therefore comprises:

-   -   (a) the chimaeric gene under the control of regulatory elements        capable of enabling the gene to be expressed when integrated in        the genome of a plant cell; and    -   (b) at least one DNA sequence which delineates the DNA to be        integrated into the plant genome.

Agrobacterium tumefaciens, therefore, containing a hybrid plasmid vectoror a binary vector in trans with a Ti-plasmid possessing a vir regioncan be used to transform plant cells. Tissue explants such as stems orleaf discs may be inoculated with the bacterium. Alternatively, thebacterium may be co-cultured with regenerating plant protoplasts. Plantprotoplasts may also be transformed by direct introduction of DNAfragments which encode the enzyme and in which the appropriatetranscriptional and translational control elements are present or by avector incorporating such a fragment. Direct introduction may beachieved using electroporation, polyethylene glycol, microinjection orparticle bombardment.

Plant cells from angiospermous, gymnospermous, monocotyledonous ordicotyledonous plants can be transformed according to the presentinvention. Monocotyledonous species include barley, wheat, maize andrice. Dicotyledonous species include cotton, lettuce, melon, pea,petunia, potato, rape, soyabean, sugar beet, sunflower, tobacco andtomato. Potato cultivars to which the invention is applicable includeDesiree, Maris Bard, Record and Russet Burbank.

Tissue cultures of transformed plant cells are propagated to regeneratedifferentiated transformed whole plants. The transformed plant cells maybe cultured on a suitable medium, preferably a selectable growth medium.Plants may be regenerated from the resulting callus. Transgenic plantsare thereby obtained whose cells incorporate the chimaeric gene in theirgenome, the chimaeric gene being expressible in the cells of the plants.Seed or other propagules from the regenerated plants can be collectedfor future use.

A preferred procedure in respect of the potato variety Record is asfollows.

Plant Material

Record shoot cultures are maintained in vitro on Murashige and Skoog(MS) medium in Magenta GA-7 containers at 22° C. (16 h/8 h light/dark).These are nodally sub-cultured every 3 weeks.

In vitro shoots of 2-3 inches (5-7.5 cm) height are potted in 2.5 inches(6.4 cm) pots of Levingtons F1 compost. They are weaned in a propagatorfor one week in a growth room at 18° C. (16 h/8 h light/dark). Thepropagator is removed and the plants repotted at 3 weeks into 5 inch(12.7 cm) pots. At 5-7 weeks the plants are used for transformation.

Agrobacterium Tumefaciens

Liquid overnight cultures of suitable strains e.g. LBA4404, C58#3 aregrown at 28° C. to an OD₆₀₀ of 0.8 in L-broth (see appendix).

Cocultivation

The youngest four most expanded leaves are taken and surface sterilisedin 10% Domestos (commercial bleach) for 15 minutes. Leaves are rinsedthoroughly with sterile water and then cut into discs with a 7 mm corkborer. The discs are mixed with the Agrobacterium for 1-5 minutes,blotted dry on filter paper (Whatman No.1) and then placed on callusingmedium (see appendix) in 90 mm triple vented petri dishes, lowerepidermis down. The 90 mm triple vented petri dishes are sealed withtape, cut to allow gas exchange and then incubated at 22° C. (16 h/8 hlight/dark). The discs are transferred to callusing medium plus 500 μgml⁻¹ of claforan and 30 μg ml⁻¹ kanamycin after 48 hours. This removesbacteria and selects for transformed cells.

Regeneration of Transformed Shoots

After 1 week, the discs are transferred to shooting medium (seeappendix) containing the same antibiotics. Further transfers are madeonto the same medium until shoots can be excised (usually about 4weeks). Shoots with calli are transferred to MS medium with cefotaximein well ventilated containers, e.g. Magenta. Transformants aremaintained, after several passages with cefotaxime to remove bacteria,on MS medium. They may be removed from in vitro, weaned and grown tomaturity as described for the stock plants.

The process yields transformed Record plants at a frequency of up to 30%of the discs cocultivated.

Appendix L-broth:  10 g l⁻¹ bacotryptone   5 g l⁻¹ yeast extract   5 gl⁻¹ sodium chloride   1 g l⁻¹ glucose Callusing medium: MS with 3%sucrose 0.5 mg l⁻¹ 2,4-D 2.5 mg l⁻¹ BAP Shooting medium: MS plus 3%sucrose 2.5 mg l⁻¹ BAP 1.0 mg l⁻¹ GA₃

The following examples illustrates the invention.

In the accompanying drawings:

FIG. 1 shows a simplified diagram of carbohydrate metabolism withreference to plant storage tissues such, for example, as potato tubers.In FIG. 1 the broken lines indicate tentatively assumed pathways.

FIG. 2 shows the procedure used to produce a chimaeric PFK gene.

FIG. 3 shows the immunodetection of E. coli PFK activity. PFK wasimmunoactivated with antisera raised to the introduced E. coli PFK.Antisera was mixed with equal amounts of PFK activity (1 nmole F6Pconsumed min ⁻¹) from two transgenic lines expressing PFK (PFK22, O;PFK8,+), two transgenic lines one not expressing PFK (PFK16*) andexpressing GUS (PS20-12), or E. coli PFK (x). Bound PFK was removed withprotein A and the activity not removed assayed (Kruger et al), Archivesof Biochemistry and Biophysics 267 690-700, 1989.

EXAMPLE 1 Production of PFK in Potato Tubers

The procedure used to produce a chimaeric PFK gene to providetuber-specific expression of PFK is illustrated in FIG. 2. The PFKcoding sequence was obtained from a clone of the pfka gene as describedby Hellinga H. W. and Evans P. R. (Eur. J. Biochem 149 363-373, 1985).The PFK coding sequence was isolated so that only 20 base pairs remainedbefore the translational start site. More specifically the E. coli pfkAgene on plasmid pHE1012 was deleted at the 5′ end to 20 bp from thetranslational start site and 50 bp from the 3′ end of the codingsequence. This was then blunt end ligated into the plasmid pFW4101 inplace of the GUS (β-glucuronidase) coding sequence to give plasmidpFW4023. pFW4101 was constructed with a patatin promoter made from twogenomic clones PS3 and PS27. The patatin fragments PS3 and PS27 werederived from the genomic clones described by Mignery et al (Gene 62,27-44, 1988). The fragments consist of −3.5 kb to −1 kb of PS3 and −1 kbto +3 kb of PS27 numbered in relation to the translation start.

E. coli harbouring pFW4023 and E. coli harbouring pFW4101 were depositedat the National Collection of Industrial and Marine Bacteria, Aberdeen,GB on 5 Jul. 1990 under accession numbers NCIMB 40305 and NCIMB 40306.

The vectors pFW4101 and pFW4023 were transferred separately intoAgrobacterium tumefaciens strain LBA 4404 by triparental mating. TheAgrobacterium strains were used to transform the potato cultivarDesiree. A large family of over 60 transgenic plants were produced.Southern analysis showed that the plants contained between one and eightcopies of the E. coli pfkA gene. Some of these plants produced tuberswhich contained considerable PFK activity. PFK activity was measured asdescribed by Kruger et al, Archives of Biochemistry and Biophysics 267690-700, 1989. Intermediates were extracted with ice cold perchlorateand measured enzymatically. The results are shown in Table 1. TABLE 1PFK Activity and amount of glycolytic intermedia PFK transgenic GUStransgenic mean (SD) mean (SD) t value P PFK activity¹ 625 (206)    29(12) 4.07 >99 Glc-6P² 78 (8.9) 100 (21) 1.97 >95 Fru-6P² 21 (4.2) 29 (9) 1.77 >90 Ratio 3.7  (0.56) 3.7    (0.66) 0.9 N.S. PEP² 82 (20.4) 28   (8.0) 3.54 >99 Pyr² 44 (22.6)  37 (16) 0.80 N.S. Ratio 2.5 (1.3)1.0   (0.6) 2.20 >95¹PFK activity is given as nmoles min⁻¹ g fr. wt.⁻¹.²Intermediates are given as nmoles g fr. wt.⁻¹.

Assays containing mixtures of extracts from two plants differing inamount of activity did not reveal the presence of activators orinhibitors (data not shown). Two lines of evidence were sought todemonstrate that the observed increase in PFK activity was due to E.coli PFK. Firstly antisera raised to this enzyme was used toimmunoinactivate specifically the E. coli PFK activity in crude proteinextracts from tubers. The results are shown in FIG. 3. A considerableproportion of the activity could be removed in lines showing increasedactivity but not in lines expressing GUS or not showing elevatedactivity (FIG. 3). Mixtures of line 12 (GUS) control plants with eitherE. coli PFK or line 22 (elevated PFK) gave the expected resultsindicating that the immunoinactivation was not due to inhibitors in thecontrol plants. Secondly the antisera was used with Western blots toshow clearly the appearance of the 36 kD E. coli PFK polypeptide of thecorrect molecular weight (data not shown). This band does not coincidewith any predominant potato protein (data not shown) or potato PFK whichhas subunit molecular weights between 55 and 63 kD.

To discover whether this increase in enzyme activity, which in thestrongest expressing tissue was 40 fold, had altered glycolytic flux weinitially measured the rate of respiration by Warburg manometry.Respiratory rates were determined by Warburg manometry (Umbreit). Tuberswere bathed in 2.7 ml of 20 mM phosphate buffer pH5.2 containing 0.5 mMglucose. CO₂ was absorbed in 10% KOH. These results are shown in Table2. TABLE 2 Respiration in Tubers Gas exchange nmol min⁻¹g⁻¹ fr.wt.(S.D.) PFK GUS Transgenic Transgenic Oxygen Uptake at 2 h 29.4 (8.9)36.3 (7.2) at 5 h 49.8 (4.1) 55.2 (10.4) CO₂ Release at 2 h 22.2 (3.2)24.3 (8.6) at 5 h 33.7 (7.7) 44.0 (9.0)

There was no indication of a change in oxygen uptake or carbon dioxideevolution. Thus if respiration determined by gas exchange is anindication of glycolytic flux, excess PFK has not altered it. However inthese tubers it is possible that a substantial amount of the respiredcarbohydrate entered the citric acid cycle via the pentose phosphatepathway and not glycolysis. Both pathways consume glucose-6-P. If thiswere the case then the addition of a large excess of PFK might changethe distribution of metabolism but not the overall flux.

We therefore determined the rate of release of ¹⁴CO₂ from 6¹⁴C-glucoseand from 1-¹⁴C-glucose. The ratio of release 6C/1C indicates thecontribution of glycolysis to respiration. In both PFK and GUStransgenic plants the ratio was approximately 0.2 after 40 mins ofincubation in ¹⁴C-glucose, 0.3 after 2 h and 0.4 after 4 h. Thus thepresence of up to 40 fold excess of PFK activity has not altered therelative contributions of glycolysis and pentose phosphate pathway toglycolytic flux.

These results suggest that PFK is not regulating the entry of carboninto glycolysis in potato tubers. We therefore measured the amounts ofglucose-6-P, and fructose-6-P, phosphoenol pyruvate (PEP) and pyruvate(Table 1). Elevated PFK activity has clearly lowered the amount ofhexose-phosphate present but the mass action ratio (Glc-6P:Fru-6P) hasremained the same and is approximately 4. This is near the equilibriumconstant of glucose-6-phosphate isomerase (Sicher and Kremer, P1.Science 67, 47-56, 1990). More notable however is the large increase inPEP and change in the ratio of PEP:pyruvate. This strongly suggests thatthe increased level of PFK has led to more carbon entering glycolysisfor a given respiratory flux and in those plants where PFK activity isincreased the enzymes (probably pyruvate kinase and PEP carboxylase)that use PEP are strongly influencing the flux.

Plants of cv Desiree transformed as-described above were grown in thefield and the amount of sucrose in the potato tubers measured at harvestwas less in lines expressing high PFK. The difference between sucrosecontent is significant at P=0.05. Thus this modification of glycolysiscan cause an alteration in a pool of metabolite in a related pathway ofcarbohydrate metabolism (as illustrated in FIG. 1). TABLE 3 Alterationin sucrose content of tubers PFK Activity nmol Sucrose content Linemin⁻¹ g⁻¹ fr.wt % w/w PFK22 1011 0.219 PFK 36 379 0.293 PS20-24 18 0.347PS20-6 18 0.358Such alterations are not confined to potato tubers. The patatin promotercan be induced to express in leaf tissue by incubating them in a mediumof sucrose (Rocha-Sosa et al (1989) EMBO J. 8 23-29). Discs were cutfrom leaves of plants (line PFK 22) containing the chimaeric PFK geneand control plants containing the chimaeric GUS gene (line PS20-12).After incubation in the light on a medium containing 1% sucrose, tocause the expression of the PFK gene, the tissues were analysed forchanges in intermediates.

The results in Table 4 show that in a tissue other than a tuber thealterations in the activity of PFK can alter metabolic intermediates.TABLE 4 $\begin{matrix}{{{Ratio}\quad{of}}\quad} \\\left( {S.D.} \right)\end{matrix}\quad\frac{\text{Amount of intermediate in line PFK-22}}{\text{Amount of intermediate in line PS20-12}}$Fru- 2,6-P₂ PEP Pyruvate 2.23 ± 0.37 1.18(±0.3) 0.49(±0.1)

EXAMPLE 2 Expression of E. coli PFK in Rice Callus

A chimaeric gene was constructed as described in FIG. 2 but a 35Spromoter replaced the patatin promoter.

This gene was used to transform rice protoplasts and the callus assayedfor PFK activity. Control callus tissue had activities of up to 1500nmol min⁻¹ g⁻¹ fr. wt. The transformed callus had activities of 3000nmol min⁻¹ g⁻¹ fr. wt. Thus it is possible to express this chimaericgene in monocotyledonous plants such as rice.

1. A process for the preparation of a transgenic plant, which methodcomprises: (i) transforming a plant cell with a chimaeric genecomprising (a) a suitable promoter and (b) a coding sequence the productof which causes modification of the amount of a metabolic intermediatein glycolysis or in a pathway for the synthesis or degradation ofstarch, sucrose or reducing sugar; and (ii) regenerating a plant fromthe transformed cell.
 2. A process according to claim 1, wherein thecoding sequence (b) encodes an enzyme selected from phosphofructokinase,pyruvate kinase, acid invertase, starch synthase, adeninediphosphoglucose, sucrose synthase, 6-pyrophofructokinase(pyrophosphate) or sucrose phosphate synthetase.
 3. A process accordingto claim 1 or 2, wherein the coding sequence (b) is from a microbialgene.
 4. A process according to claim 3, wherein the coding sequence (b)is from a bacterial gene.
 5. A process according to claim 1, wherein thecoding sequence (b) encodes for phosphofructokinase.
 6. A processaccording to claim 1, wherein the coding sequence (b) encodes for two ormore enzymes.
 7. A process according to claim 1, wherein the plant celltransformed in step (i) is a cell of (A) a monocotyledonous speciesselected from the group consisting of barley, wheat, maize and rice; or(B) a dicotyledonous species selected from the group consisting ofcotton, lettuce, melon, pea, petunia, potato, rape, soyabean, sugarbeet, sunflower, tobacco and tomato.
 8. A process according to claim 7,wherein the plant cell transformed is a potato cultivar cell, saidcultivar is selected from the group consisting of Desiree, Maris Bard,Record and Russet Burbank.
 9. A chimaeric gene as defined in claim 1.10. A vector which comprises a chimaeric gene as defined in claim 1 suchthat the chimaeric gene is capable of being expressed in a plant celltransformed with the vector.
 11. A vector according to claim 10, whichis a plasmid.
 12. A plant cell which harbours a chimaeric gene asdefined in claim 1 such that the chimaeric gene is capable of beingexpressed therein.
 13. A transgenic plant which harbors in its cells achimaeric gene as defined in claim 1 such that the chimaeric gene iscapable of being expressed in the cells of the plant.
 14. A plantaccording to claim 13, which is a potato.
 15. Seed obtained from atransgenic plant as claimed in claim 13 or
 14. 16. Tubers obtained froma potato as claimed in claim 14.